The use of synthetic phosphodiester oligonucleotides in living cells faces two major challenges: 1) rapid degradation of the compounds in vivo and 2) low uptake and inefficient transport through plasma membranes. To overcome these problems, various approaches have been tested in recent decades. One of the successful strategies has been synthesis of chemically modified oligonucleotides, particularly chemical modification of the backbones of oligonucleotides, insofar as this approach provides preservation of Watson-Crick hybridization properties of compounds. Synthetic oligonucleotides with phosphorothioate (Padmapriya et al., (1994) Antisense Res. Dev., 4, 185-199), methylphosphonate (Reddy et al., (1996) Tetrahedron Lett., 37, 8691-8694), boronophosphate (Shaw et al., (1993) Methods Mol. Biol., 20, 225-243), benzylphosphonate (Samstag et al., (1996) Antisense Nucleic Acids Drug Dev., 6, 153-156) modifications, locked nucleic acid (Kurreck et al., (2002) Nucleic Acids Res., 30, 1911-1918; Crinelli et al., (2002) Nucleic Acids Res., 30, 2435-2443), peptide nucleic acid (Egholm et al., (1993) Nature, 365, 566-568), morpholino derivatives (Stirchak et al., (1989) Nucleic Acids Res., 17, 6129-6141; Sinha et al., (1984) Nucleic Acids Res., 12, 4539-4557), and oligonucleotides with modified terminal groups (Shchepinov et al., (1997) Nucleic Acids Res., 25, 4447-4454; Shchepinov et al., (1999) Nucleic Acids Res., 27, 3035-3041; Horn et al., (1997) Nucleic Acids Res., 25, 4842-4849; Guzaev et al., (1995) Tetrahedron, 51, 9375-9384) have been widely tested as novel therapeutic agents (Monteith et al., (1999) Toxicol. Pathol., 27, 8-13; Ma et al., (2000) Biotechnol. Ann. Rev., 5, 155-196; Wilson and Richardson, (2006) Infect. Disord. Drug Targets, 6, 43-56) and diagnostic tools (Landegren et al., (1988) Science, 242, 229-237).
For some applications, native or modified synthetic oligonucleotides have to be linked to non-nucleotide molecules or surfaces. The non-nucleotide molecules could be fluorescent dyes or quenchers, metal chelators, ligands for various proteins, enzymes, receptors, transporters, or other biologically active molecules, hydrophobic residues, or even other oligonucleotides. The resultant conjugates may be useful as hybridization probes in DNA sequencing and microarray technology, as diagnostic and therapeutic agents, electron and fluorescent microscopy probes, and have roles in crystallography, affinity chromatography, and in cell biology research (Goodchild et al., (1990) Bioconjugate Chem., 1; 165-187; DaRos et al., (2005) Curr. Med. Chem. 12, 71-88; Boutorine et al., (2000) Molecular Biology, 34, 804-813; Urban and Noe, (2003) Farmaco, 58, 243-258; Silverman and Kool, (2006) Chem. Rev., 106, 3775-3789). Because oligonucleotides, with the exception of phosphorothioates, lack appropriately reactive functional groups for conjugate synthesis, introduction of such groups is necessary.
One of the most convenient and widely used groups for conjugation purposes is the aliphatic amino group. It can be coupled selectively and under mild conditions with ligands (reporters) bearing carboxylic groups or their activated derivatives, sulfonyl chlorides, isocyanates and isothiocyanates, aldehydes and alkylating residues, or other electrophilic functionalities. The amino group is usually attached to the oligonucleotide by a linker (spacer). The nature and the length of the linker are important for the synthesis and the function of the conjugates. Short linkers can create spatial restrictions, which lower the reactivity of the amino group and interfere with the function of the ligand and the oligonucleotide. Hydrophobic linkers, even when of sufficient length, tend collapse in aqueous environments, assuming globular conformations and again creating spatial crowding.
The site of the spacer attachment is another important factor in determining the properties of the conjugates. Traditionally, the spacer is attached at the 5′-end of the oligonucleotide upon the completion of the automated synthesis (Tang and Agrawal, (1990) Nucleic Acids Res., 18, 6461; Agrawal and Zamecnik, (1990) Nucleic Acids Res., 18, 5419-5423; Agrawal et al., (1986) Nucleic Acids Res., 14, 6227-6245; Kachalova et al., (2002) Helv. Chim. Acta., 85, 2409-2417). The attachment to the 3′-end on non-standard supports has also been explored (Markiewicz et al., (1997) Nucl. Acids Res., 25, 3672-3680). One drawback of both methods is that they do not allow position-specific modifications. If multiple reporters are to be attached, they cluster at the end of the oligonucleotide with no convenient strategy to vary their relative positions. When a reporter is to be placed within a certain sequence, or when multiple reporters are to be placed within a desired distance, intrastrand modification is necessary. The standard approach is to attach a linker to C-5 of the pyrimidine bases or C-8 of the purines. Deoxyribonucleoside phosphoramidites of this type with trifluoroacetyl-protected amino groups are commercially available. Thus, the design of the spacer and the place of its attachment in those synthons are driven more by consideration of synthetic convenience than by functionality. In the case of purines, an aliphatic spacer is attached to C-8 through an amino group, and in the case of pyrimidines, a carbamoylvinyl group introduced at C-5 is in conjugation with the base. Both alternatives could potentially result in interfering with normal base pairing.
The attachment of a linker to the internucleoside phosphates enables sequence-specific and multi-site labeling, minimally perturbs Watson-Crick base pairing and, importantly, provides stabilization of the oligonucleotides toward nucleases (Wenninger et al., (1998) Nucleos. Nucleot., 17: 2117-2125; Awad et al., (2004) Nucleosides, Nucleotides & Nucleic Acids, 23, 777-787). Two major types of internucleoside modifications have been described: 1) using a spacer linked to the sulphur of phosphorothioates, and 2) phosphoramidate nitrogen-linked spacers (Agrawal and Zamecnik, (1990) Nucleic Acids Res., 18, 5419-5423). The first type of linker is generally prepared by alkylation of phosphorothioates, and the second by oxidation of H-phosphonates in the presence of 1,ω-diamines. Drawbacks of these strategies include incompatibility with the commonly used phosphoramidite-based automated oligonucleotide synthesis, and they may require additional post-synthetic modifications.
The attachment of a linker to the oxygen of the internucleoside phosphates has been explored using a sequence-specific attachment of 2-aminoethyl groups (Seliger et al., (1991) Nucleos. Nucleot. 10, 303-306). However, the short 2-3 carbon atom spacer is not optimal for further attachment of reporters or ligands to the amino group. Increasing the length of the spacer results in its destabilization, because of the intramolecular attack at the terminal amino group of carbon atom adjacent to the phosphate through a favorable five or six atom cyclic transition state, resulting in the spacer's scission. A 4-trifluoroacetamidobutyl group has been developed as an alternative to the cyanoethyl group for phosphate protection in oligonucleotide synthesis (Wilk et al., (1999) J. Org. Chem., 64, 7515-7522).